The use of immunoconjugates for the selective delivery of cytotoxic agents to tumor cells in the treatment of cancer is known in the art. The delivery of cytotoxic agents to the site of tumor cells is much desired because systemic administration of these agents often results in the killing of normal cells within the body as well as the tumor cells sought to be eliminated. Thus, according to the antitumor drug delivery systems currently in use, a cytotoxic agent is conjugated to a tumor-specific antibody to form an immunoconjugate that binds to the tumor cells and thereby "delivers" the cytotoxic agent to the site of the tumor. The immunoconjugates utilized in these targeting systems include antibody-drug conjugates [see, e.g., R. W. Baldwin et al., "Monoclonal Antibodies For Cancer Treatment," Lancet, pp. 603-05 (Mar. 15, 1986)] and antibody-toxin conjugates [see, e.g., P. E. Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506 (1985)].
Both polyclonal antibodies and monoclonal antibodies have been utilized in these immunoconjugates [see, e.g., K. Ohkawa et al., "Selective In Vitro And In Vivo Growth Inhibition Against Human Yolk Sac Tumor Cell Lines By Purified Antibody Against Human .alpha.-Fetoprotein Conjugated With Mitomycin C Via Human Serum Albumin," Cancer Immunol. Immunother., 23, pp. 81-86 (1986) and G. F. Rowland et al., "Drug Localisation And Growth Inhibition Studies Of Vindesine-Monoclonal Anti-CEA Conjugates In A Human Tumour Xenograft," Cancer Immunol. Immunother., 21, pp. 183-87 (1986). Drugs used in these immunoconjugates include daunomycin [see, e.g., J. Gallego et al., "Preparation Of Four Daunomycin-Monoclonal Antibody 791T/36 Conjugates With Anti-Tumour Activity," Int. J. Cancer, 33, pp. 737-44 (1984) and R. Arnon et al., "In Vitro And In Vivo Efficacy Of Conjugates Of Daunomycin With Anti-Tumor Antibodies," Immunological Rev., 62, pp. 5-27 (1982)], methotrexate [N. Endo et al., "In Vitro Cytotoxicity Of A Human Serum Albumin-Mediated Conjugate Of Methotrexate With Anti-MM46 Monoclonal Antibody," Cancer Research, 47, pp. 1076-80 (1987)], mitomycin C [K. Ohkawa et al., supra], and vindesine [G. F. Rowland et al., supra]. Toxins used in the antibody-toxin conjugates include bacterial toxins such as diptheria toxin and plant toxins such as ricin [see, e.g., F. L. Moolten et al., "Antibodies Conjugated To Potent Cytotoxins As Specific Antitumor Agents," Immunol. Rev., 62, pp. 47-73 (1982)].
Despite the amount of research directed towards the use of immunoconjugates for therapeutic purposes, several limitations involved with these delivery approaches have become apparent [see, e.g., M. J. Embleton, "Targeting Of Anti-Cancer Therapeutic Agents By Monoclonal Antibodies," Biochemical Society Transactions 14, pp. 393-395 (615th Meeting, Belfast 1986)]. Firstly, the large amount of drug required to be delivered to the target tumor cell to effect killing of the cell is often unobtainable because of limitations imposed by the number of tumor-associated antigens on the surface of the cells and the number of drug molecules that can be attached to any given antibody molecule. This limitation has led to the use of more potent cytotoxic agents such as plant toxins in these conjugates and to the development of polymer-bound antibody-drug conjugates having very high drug multiplicity ratios [see, e.g., P. E. Thorpe, supra, pp. 475-506 and R. W. Baldwin et al., "Design And Therapeutic Evaluation Of Monoclonal Antibody 791T/36 --Methotrexate Conjugates," in Monoclonal Antibodies And Cancer Therapy, pp. 215-31 (Alan R. Liss, Inc. 1985)]. However, even with large drug loading ratios or with the use of potent toxins, many immunoconjugates still display sub-optimal cytotoxic activity and are unable to effect complete killing at doses where all available antigenic sites are saturated.
Secondly, it has been recognized that the cytotoxic activity of an immunoconjugate is often dependent on its uptake, mediated by the antibody component of the conjugate, into the tumor cell [see, e.g., J. M. Lambert et al., "Purified Immunotoxins That Are Reactive With Human Lymphoid Cells," J. Biol. Chem., 260 (No. 22), pp. 12035-12041 (1985)]. This internalization is crucial when using an antibody-drug conjugate in which the drug has an intracellular site of action or when using antibody-toxin conjugates. However, the vast majority of tumor-associated antigens and thus the antibody-drug or antibody-toxin conjugates bound to those antigens, are not internalized. Those conjugates that are internalized are often transported to the lysosome of the cell where the drug or toxin is degraded [see, E. S. Vitetta et al., Science. 238, pp. 1098-1104 (1987)]. Accordingly, although an antibody-drug or antibody-toxin conjugate may have excellent tumor-binding characteristics, the conjugate may nonetheless have a limited cytotoxic utility due to an inability to reach its site of action within the cell.
In addition, it is well established that tumor cell populations are often heterogeneous with respect to antigen expression [see, e.g., A. P. Albino et al., "Heterogeneity In Surface Antigen And Glycoprotein Expression Of Cell Lines Derived From Different Melanoma Metastases Of The Same Patient," J. Exp. Med., 154, pp. 1764-78(1981)]. Furthermore, it has been demonstrated that antigen-positive tumor cells may give rise to antigen-negative progeny [see, e.g., M. Yeh et al., "Clonal Variation In Expression Of A Human Melanoma Antigen Defined By A Monoclonal Antibody," J. Immunol., 126 (No. 4), pp. 1312-17 (1981)]. Thus, in any population of tumor cells, there will be a certain number of cells that do not possess the antigen for which a particular immunoconjugate is specific. The immunoconjugate will therefore not be able to bind to these cells and mediate their killing.
Due to these drawbacks, the currently utilized antitumor drug or toxin delivery systems have had a limited amount of success, especially when used for in vivo treatment.
In addition to the immunoconjugates discussed above, antibody-enzyme conjugates have been studied in vitro in combination with a second untargeted enzyme for the conversion of iodide or arsphenamine to their toxic forms in order to amplify antibody-mediated cytotoxicity [see, e.g., C. W. Parker et al., "Enzymatic Activation And Trapping Of Luminol-Substituted Peptides And Proteins. A Possible Means Of Amplifying The Cytotoxicity Of Anti-Tumor Antibodies," Proc. Natl. Acad. Sci. USA, 72 (No. 1), pp. 338-42 (1975) and G. W. Philpott et al., "Affinity Cytotoxicity Of Tumor Cells With Antibody-Glucose Oxidase Conjugates, Peroxidase, And Arsphenamine," Cancer Research, 34, pp. 2159-64 (1974)].
According to these in vitro studies, the enzyme, glucose oxidase, is attached to an antibody and used in combination with an untargeted peroxidase enzyme to convert iodide or arsphenamine to cytotoxic iodine or arsenical, respectively. This approach, therefore, requires not only the targeting of glucose oxidase to tumor cells with antibody, but also the presence at the tumor site of two other untargeted agents. The likelihood that all three of these agents will be present in vivo at the tumor site at the same time is small and therefore this approach is unlikely to be of therapeutic importance.
Canadian patent No. 1,216,791, issued to F. Jansen et al., on Jan. 20, 1987, discloses the conjugation to an antibody of an enzyme capable of liberating ammonium ions from substrates. The ammonium ions are then said to potentiate the cytotoxic action of certain immunotoxins targeted to the tumor site.
Finally, European patent application No. 84302218.7 discloses a method for treating a diseased cell population such as a tumor wherein an antibody is used to target a non-metabolizable antigen to the tumor cells. The antigen accumulates within at least a percentage of the tumor cells, which are then lysed to release the antigen into a ubiquitous fibronectin capturing matrix formed at the tumor site. At this point in the method of the invention, an iodine-containing ligand which is specific for and will bind to the antigen affixed to the matrix is administered. The cytotoxic iodine then acts to kill the tumor cells at that site. Many alternative embodiments are disclosed in this application, one of which suggests the use of an antibody-enzyme conjugate to target enzyme to a tumor site and the addition of a non-lethal substrate which the enzyme can convert to a cytotoxic material [see European application, pp. 34-35]. However, nowhere in the application is there any disclosure of how one is to perform this embodiment. Similarly, Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson and Lee (ed.s), p. 639(1987) suggest that "[d]rugs which would be non-toxic until `activated` by an agent (e.g., an enzyme) localized to tumor may be considered as another approach. . . . "
To date, however, no one has disclosed or suggested how the approach provided herein might be carried out nor has anyone actually attempted this approach to drug targeting.